1. Field of the Invention
The present invention is directed to methods for identifying compounds that modulate apoptosis, directly or indirectly, as measured by changes in expression of the bbc3 gene or a reporter gene, or by changes in the apoptotic character of a cell.
The present invention is also directed to methods for identifying compounds that modulate expression of the bbc3 gene and a method for identifying compounds that interact with a promoter of the bbc3 gene.
The present invention is further directed to a method for modulating apoptosis in a cell, a method for modulating bbc3 gene expression in a cell, a process for making a modulator of apoptosis, and a process for making a modulator of bbc3 gene expression.
Finally, the present invention is directed to compounds identified by each of the methods of this invention, and to promoters of the bbc3 gene and sequences homologous thereto.
2. Description of the Related Art
Bcl-2 family proteins regulate the cellular response to apoptotic stimuli (Adams, J. M., et al., Science 281:1322–6 (1998); Korsmeyer, S. J., Cancer Res. 59:1693s–1700s (1999)). Members of this family can function to either suppress or promote cell death, and are characterized by the presence of up to four different conserved amino acid motifs, termed Bcl-2 homology (BH) domains (Adams, J. et al., Science 281:1322–6 (1998); Korsmeyer, S. J., Cancer Res. 59:1693s–1700s (1999)). The BH3 domain is a uniquely important functional element within the pro-apoptotic class of Bcl-2 related proteins, mediating the ability of these proteins to dimerize with other Bcl-2 related proteins, and promote apoptosis (Chittenden, T., et al., Nature 374:733–6 (1995); Huang, D. C., et al., Cell 103:839–42 (2000)). The function of BH3 as a cell death domain has been further revealed by an emerging group of ‘BH3-only’ proteins, which share only BH3 in common with other Bcl-2 family members. This class of proteins, which includes Bik, Bad, Bid, Bim, Egl-1 and Hrk, induce apoptosis by binding, through the BH3 domain, to anti-apoptotic members of the Bcl-2 family (Huang, D. C., et al., Cell 103:839–42 (2000)).
Biochemical and genetic evidence indicates that BH3-only proteins function at a point upstream of Bcl-2 in a cell death pathway conserved in both vertebrates and invertebrates (Huang, D. C., et al., Cell 103:839–42 (2000); Horvitz, H. R., Cancer Res. 59:1701s–1706s (1999)). BH3-only proteins have been shown to localize to mitochondria following apoptotic stimuli, where they bind to Bcl-2 family members and induce mitochondrial events associated with apoptosis, including the release of cytochrome c into the cytosol (Huang, D. C., et al., Cell 103:839–42 (2000)). The functions of the BH3 proteins Bad and Bid are regulated by phosphorylation and proteolytic activation, respectively, in response to extrinsic cell survival/cell death stimuli (Zha, J., et al., Cell 87:619–28 (1996); Luo, X., et al., Cell 94:481–90 (1998); Li, H., et al., Cell 94:491–501 (1998)). Through post-translational modifications, Bad and Bid transduce signals originating at cell surface receptors to a Bcl-2-regulated, mitochondrial apoptosis control point.
In C. elegans, the BH3-only protein Egl-1 operates at the most proximal point in a genetically-defined pathway required for all programmed cell death (Conradt, B., et al., Cell 93:519–29 (1998)). Egl-1 binds to the nematode Bcl-2 counterpart, Ced-9, and antagonizes its function, similar to the function ascribed to mammalian BH3 proteins. By contrast to Bad and Bid, the activity of Egl-1 appears to be regulated primarily through transcriptional control mechanisms. The egl-1 gene is active specifically in cells that are destined to die during development, and genetic studies have identified transcription factors upstream of egl-1 that control its expression in certain cell lineages (Conradt, B., et al., Cell 98:317–27 (1999); Metzstein, M. M., et al., Nature 382:545–7 (1996); Metzstein, M. M., et al., Mol. Cell. 4:309–19 (1999)).
There is a remarkable degree of structural and functional conservation between the genes that control the cell death pathway in nematodes and mammals (Horvitz, H. R., Cancer Res. 59:1701s–1706s (1999)). The essential contribution of Egl-1 and its transcriptional regulation to programmed cell death in C. elegans strongly implies that apoptosis in mammalian cells may depend on the transcriptional control of gene(s) encoding BH3-only proteins. Disregulation of the mechanisms that control transcription of BH3-only genes in this class may contribute to defects in apoptosis in diseases such as cancer.
The present inventors recently identified a strongly pro-apoptotic BH3-only protein, termed Bbc3 (Bcl-2 binding component 3), encoded by a gene (bbc3) (the human bbc3 cDNA, promoter and protein sequences can be found at GenBank accession numbers U82987, AF411827 and AAB51243, respectively) that is subject to transcriptional regulation by multiple cell death signaling pathways. Experimental results have indicated that transcriptional control of the bbc3 gene contributes to cell death regulation in mammalian cells. Bbc3 gene expression is activated by at least three apoptotic stimuli, including DNA damage, glucocorticoid treatment, and growth factor deprivation, constituting a broad transcriptional response thus far unique among mammalian cell death regulatory genes. By analogy to egl-1, the bbc3 gene may prove to be induced in other contexts as well, for example, during developmental cell deaths. Thus, expression of the bbc3 gene may serve to transduce cell death signals originating from diverse stimuli to common mitochondrial apoptotic events regulated by the Bcl-2 family.
Due to the apparent broad role of Bbc3 in pathways leading to apoptosis, the identification of compounds that modulate the effects of Bbc3 could lead to the development of therapeutic agents. Indeed, the identification of compounds that modulate bbc3 gene expression, and those that modulate activity of the Bbc3 protein, could potentially be used in the treatment of medical conditions such as cancers where the normal apoptotic pathways have been blocked (e.g. leukemia), and neuronal degenerative diseases, such as Parkinson's disease, where apoptosis is accelerated.
In addition, the bbc3 gene could be used to identify components of the bbc3 signal transduction pathway, as well as components of other pathways involved in apoptosis. Compounds that modulate these pathways could also be important therapeutic agents useful for treating a range of medical conditions.
Accordingly, methods of identifying compounds that modulate both apoptosis and the activity of the bbc3 gene could serve to identify potential therapeutic compounds, as well as identify other targets for treatment of medical conditions where apoptosis is involved. The present invention is directed to these and other important objectives.